1. Field of the Invention
The present invention is broadly concerned with improved methods for the forming and precipitation of small protein or peptide particles making use of the precipitation using compressed antisolvents (PCA) process. More particularly, the invention is concerned with such a method and the resulting proteinaceous particles wherein the process is carried out using a halogenated organic alcohol as at least a part of the protein solvent; in particularly preferred forms, the solvent is 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP), and the process yields micron-sized particles suitable for pharmaceutical uses without substantially degrading the protein.
2. Description of the Prior Art
Micron-sized (1-10 xcexcm) protein particles are often deemed necessary for drug delivery systems such as controlled release and direct aerosol delivery to the lungs. Consistent commercial production of small protein particles of this type can be difficult. For example, spray drying techniques often lead to thermal denaturation of the protein, while milling and similar processes yield unacceptably broad size distributions and/or denaturation. Lyophilization can give particles in the desired size range, but with a broad distribution and/or denaturation; moreover, not all proteins of interest can be lyophilized to stable products.
In an effort to overcome these problems, supercritical fluid precipitation processes have been employed. Two processes that use supercritical fluids for particle formation are: (1) Rapid Expansion of Supercritical Solutions (RESS) (Tom, J. W. Debenedetti, P. G., 1991, The Formation of Bioerodible Polymeric Microspheres and Microparticles by Rapid Expansion of Supercritical Solutions. BioTechnol. Prog. 7:403-411), and (2) Gas Anti-Solvent (GAS) Recrystallization (Gallagher, P. M., Coffey, M. P., Krukonis, V. J., and Klasutis, N., 1989, Gas Antisolvent Recrystallization: New Process to Recrystallize Compounds in Soluble and Supercritical Fluids. Am. Chem. Sypm. Ser., No. 406; U.S. Pat. No. 5,360,478 to Krukonis et al.; U.S. Pat. No. 5,389,263 to Gallagher et al.). See also, PCT Publication WO 95/01221 and U.S. Pat. No. 5,043,280 which describe additional SCF particle-forming techniques.
In the RESS process, a solute (from which the particles are formed) is first solubilized in supercritical CO2 to form a solution. The solution is then sprayed through a nozzle into a lower pressure gaseous medium. Expansion of the solution across this nozzle at supersonic velocities causes rapid depressurization of the solution. This rapid expansion and reduction in CO2 density and solvent power leads to supersaturation of the solution and subsequent recrystallization of virtually contaminant-free particles. The RESS process, however, is not suited for particle formation from polar compounds because such compounds, which include drugs, exhibit little solubility in supercritical CO2. Cosolvents (e.g., methanol) may be added to CO2 to enhance solubility of polar compounds; this, however, affects product purity and the otherwise environmentally benign nature of the RESS process. The RESS process also suffers from operational and scale-up problems associated with nozzle plugging due to particle accumulation in the nozzle and to freezing of CO2 caused by the Joule-Thompson effect accompanying the large pressure drop.
The relatively low solubilities of pharmaceutical compounds in unmodified carbon dioxide are exploited in the second process wherein the solute of interest (typically a drug, polymer or both) is dissolved in a conventional solvent to form a solution. The preferred ternary phase behavior is such that the solute is virtually insoluble in dense carbon dioxide while the solvent is completely miscible with dense carbon dioxide at the recrystallization temperature and pressure. The solute is recrystallized from solution in one of two ways. In the first method, a batch of the solution is expanded several-fold by mixing with dense carbon dioxide in a vessel. Because the carbon dioxide-expanded solvent has a lower solvent strength than the pure solvent, the mixture becomes supersaturated forcing the solute to precipitate or crystallize as microparticles. This process was termed Gas Antisolvent (GAS) recrystallization (Gallagher et al., 1989).
The second method involves spraying the solution through a nozzle into compressed carbon dioxide as fine droplets. In this process, a solute of interest (typically a drug, polymer or both) that is in solution or is dissolved in a conventional solvent to form a solution is sprayed, typically through conventional spray nozzles, such as an orifice or capillary tube(s), into supercritical CO2 which diffuses into the spray droplets causing expansion of the solvent. Because the CO2-expanded solvent has a lower solubilizing capacity than pure solvent, the mixture can become highly supersaturated and the solute is forced to precipitate or crystallize. This process has been termed in general as Precipitation with a Compressed Fluid Antisolvent (PCA) (Dixon, D. J.; Johnston, K. P.; Bodmeier, R. A. AIChE J. 1993, 39, 127-139.) and employs either liquid or supercritical carbon dioxide as the antisolvent. When using a supercritical antisolvent, the spray process has been termed Supercritical Antisolvent (SAS) Process (Yeo, S.-D.; Debenedetti, P. G.; Radosz, M.; Schmidt, H.-W. Macromolecules 1993, 26, 6207-6210.) or Aerosol Spray Extraction System (ASES) Mxc3xcller, B. W.; Fischer, W.; Verfahren zur Herstellung einer mindestens einen Wirkstoff und einen Trxc3xa4ger umfassenden Zubereitung, German Patent Appl. No. DE 3744329 A1 1989).
U.S. Pat. No. 6,063,910 describes a specific process for the production of protein particles by supercritical fluid precipitation. In this process, a solution of protein is prepared using a variety of solvents such as ethanol, DMSO and glycols, whereupon the solution is sprayed through a nozzle into an antisolvent under supercritical conditions, thereby effecting precipitation of the protein as small micron-sized products. In the case of insulin, the process was found to create a substantial loss of xcex1-helicity and a marked increased in xcex2-sheet and xcex2-reverse turn content. (Winters et al., J. Pharmaceutical Sciences, 85(6):586-594 (1996)). The solvents used in the process of the ""910 patent, and particularly DMSO, are not favored for pharmaceutical uses. For example, many such solvents leave a residuum in the precipitated particles, causing purity problems.
There is accordingly a real and unsatisfied need in the art for an improved protein precipitation process which avoids the solvent problems of many prior techniques while giving micron-sized particles of small size distribution and with little protein degradation.
The present invention overcomes the problems outlined above and provides an improved method for forming small protein particles of micron size, preferably from about 1-10 xcexcm, and more preferably from about 1-5 xcexcm. Broadly speaking, the process involves contacting a protein, a protein solvent system and an antisolvent for the protein solvent system under conditions to at least partially dissolve the protein solvent system in the antisolvent with consequent precipitation of the protein; the protein solvent system includes at least in part a halogenated organic alcohol, and preferably consists essentially of a single halogenated organic alcohol. Use of such solvents materially improves precipitation processes heretofore used and avoids many of the problems of the prior art.
A wide variety of solvent/antisolvent precipitation processes can be used in accordance with the invention. For example, the GAS and PCA processes can be employed. Preferably however, the solvents of the invention are used in the PCA process wherein the protein is first dissolved in the solvent system, and then droplets of the solution are sprayed into an antisolvent under conditions to precipitate protein particles.
The preferred halogenated organic alcohols are the halogenated alkyl alcohols, especially the C1-C4 alcohols. Particular alcohol solvents are HFIP, trifluoroethanol, 2-chloroethanol and mixtures thereof. The single most preferred solvent is HFIP (CAS #920-66-1). This solvent has a boiling point of 59xc2x0 C. and a density of 1.618 g/ml, and is very soluble in CO2. Normally, only a single halogenated organic alcohol will be used as a protein solvent. However, multiple-component solvent systems can also be employed, so long as such systems include a halogenated organic alcohol as at least a part thereof.
A variety of antisolvents can also be used in the invention, such as CO2, propane, butane, isobutane, nitrous oxide, sulfur hexafluoride, trifluoromethane, hydrogen and mixtures thereof. CO2 is the most preferred antisolvent, owing to its low cost, ready availability and critical properties (Tc=81.0xc2x0 C. and Pc=73.8 bar or 1070 psi). Furthermore, CO2 is non-toxic, non-flammable, recyclable, and xe2x80x9cgenerally regarded as safexe2x80x9d by the FDA and pharmaceutical industry.
During processing, the contact between the protein solution system and antisolvent is carried out at near or supercritical conditions for the antisolvent, e.g., from about 0.5-2 Pc and more preferably from about 0.9-1.5 Pc; when CO2 is used as the antisolvent, pressure conditions are normally maintained at a level of from about 1000-2000 psig, and more preferably from about 1100-1600 psig. The temperature conditions during processing are generally relatively low in order to avoid heat denaturation of the protein. Generally, temperatures of up to about 60xc2x0 C. and more preferably up to about 50xc2x0 C. are used. When CO2 is the antisolvent, such temperatures exceed the Tc.
In order to maximize production rates, the preferred process is carried out in a pressurized precipitation chamber equipped with a nozzle. The protein solution is sprayed through the nozzle into a precipitation zone containing the antisolvent. The resultant protein particles are collected in a downstream recovery filter, and can easily be further processed for pharmaceutical uses.
In most instances, the starting protein is dissolved in a halogenated organic alcohol solvent or solvent system containing such an alcohol, thereby producing true solutions. However, the invention is not so limited. That is, it is possible that the protein may be only partially dissolved or dispersed within the solvent. Therefore, as used herein, xe2x80x9csolutionxe2x80x9d should be understood to mean not only true solutions but also partial solutions and dispersions. Similarly, while complete proteins are often processed in accordance with the invention, protein fragments or peptides could also be treated. Accordingly, the term xe2x80x9cproteinxe2x80x9d refers to all types of proteinaceous species.